Tissue processing reagent

ABSTRACT

A method including placing a biological sample taken from a body into a chamber; adding a composition including an acetal solvent to the chamber; and fixating the biological sample. A method including placing a tissue from a body into a chamber; and contacting the tissue with a composition including an acetal solvent as a fixating process.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/371,588 titled “Tissue Processing Reagent,” filed Dec. 7, 2016, nowU.S. Pat. No. 10,107,727 issued on Oct. 23, 2018, which is acontinuation-in-part of U.S. patent application Ser. No. 14/984,946,titled “Tissue Processing Reagent,” filed Dec. 30, 2015, now U.S. Pat.No. 9,835,527 issued on Dec. 5, 2017, the entire contents of which areincorporated herein by reference.

FIELD

Tissue processing.

BACKGROUND

Tissues from the body taken for diagnosis of disease processes are oftenprocessed in the histology laboratory to produce thin tissue sectionswhich can be mounted on slides and viewed under a microscope by apathologist for analysis. These pre-analytical processes generallyinclude, in order, gross examination fixation, dehydration, clearing,paraffin infiltration and embedding. The procedure is used forprocessing tissues including biopsies, larger specimens removed atsurgery, or tissues from autopsy.

Gross examination generally consists of describing the macroscopicspecimen and placing all or selected parts of it into a small plasticcassette which holds the tissue while it is being processed to aparaffin block. Initially, the cassettes are placed into a fixative.

Following gross examination, a tissue is fixated. A purpose of fixationis to preserve tissues permanently in as life-like a state as possibleby altering structures of proteins such that degradation by autolysisdoes not occur. A variety of fixatives are available for use, dependingon the type of tissue present and features to be demonstrated. Majorgroups of fixatives, classified according to mechanism of action includealdehydes, mercurials, alcohols, oxidizing agents and picrates. Formalinfixation is best carried around neutral pH, for example, in the range of6-8. Hypoxia of tissues tends to lower the pH, so there should bebuffering capacity in the fixative to prevent excessive acidity. Commonbuffers include phosphate, bicarbonate, malate, cacodylate, and veronal.Commercial formalin, for example, may be buffered with phosphate at a pHof 7. Penetration of tissues depends upon the diffusability of eachindividual fixative. One way to improve penetration of a fixative is togross (cut) the tissue thinly (2 to 3 millimeters (mm)). Penetrationinto a thin tissue section will occur more rapidly than for a thicksection. The volume of fixative is generally important with a 10:1 ratioor greater of fixative to tissue typically targeted. Agitation of thespecimen in a fixative will often also enhance fixation.

Once the tissue has been fixed or fixated, the tissue needs to beprocessed into a form in which it can be made into thin sections formicroscopic examination. The usual way this is done is with paraffin.Tissues embedded in paraffin, which provides a solid support matrix forthe tissue, allowing it be sectioned at a thickness on the order of 2 to20 microns. Getting fixed tissue into paraffin for sectioning is calledtissue processing with the main steps in this process being dehydration,clearing, infiltration and embedding.

Tissues fixed in aqueous solutions cannot be directly infiltrated withparaffin. First, the water from the tissues must be removed bydehydration. This may be done with a series of alcohols at differentconcentrations (e.g., 70 percent to 95 percent to 100 percent).Alternatively, the dehydration is done with a mixture of formalin andalcohol. Other dehydrants can also be used such as acetone or mixturesof different solvents.

Following dehydration, the tissue is cleared. “Clearing” consists ofremoval of the dehydrant and some of the lipids with a substance thatwill be miscible with the embedding medium (e.g., paraffin). The mostcommon clearing agent is xylene.

Once cleared, the tissue is infiltrated with an embedding agent such asparaffin. Finally, the tissue in a cassette or removed from its cassetteis placed into molten paraffin and then the paraffin is cooled to form asolidified block embedding or encapsulating the tissue so that it can besectioned. Alternatively, the tissue can be processed in a sectionablecassette, embedded in paraffin along with the cassette and sectioned.Once the tissue has been embedded in a solid paraffin block, the tissuecan be cut into sections that can be placed on a slide. This is donewith a microtome. Once sections are cut, they are floated on a warmwater bath that helps remove any wrinkles. The tissue sections inparaffin are then picked up from the water bath and placed on a glassmicroscope slide.

DETAILED DESCRIPTION

In one embodiment, a composition including an acetal solvent isdisclosed that is operable or suitable for treating a biological sampletaken from a body. A biological sample such as a tissue taken from thebody for diagnosis or research includes but is not limited to, a biopsy,a specimen removed at surgery and/or tissues from autopsy. Alsodisclosed is a method including treating a biological sample taken froma body for diagnosis with a composition including an acetal. Treating abiological sample in this regard, in one embodiment, is focused on apre-analytical process such as fixation, clearing and/or embedding ofthe biological sample for subsequent examination/diagnosis.

Representative acetals suitable for a composition for treating abiological sample include methylal, ethylal, butylal, dioxolane,glycerol formal, acetaldehyde diethyl acetal and mixtures thereof.Dioxolane is one particular preferred acetal solvent. A dioxolane asdescribed herein includes 1,3-dioxolane as well as its adducts andmixtures thereof. Such adducts include, but are not limited, to2-methyl-1,3-dioxolane; 4-methyl-1,3-dioxolane;2,2-dimethyl-1,3-dioxolane; 2-methyl-1,3-dioxolane;4-methyl-2-phenyl-1,3-dioxolane (benzaldehyde propylene glycol),1,2-dioxolane and adducts thereof. 1,3-dioxolane is one particularpreferred dioxolane due to its favorable toxicity profile and commercialavailability.

In one embodiment, a composition including a dioxolane is used in aprocess in the histology laboratory to produce microscopic slides thatcan be viewed under a microscope for analysis. In one embodiment, acomposition including a dioxolane can be used as a dehydrating agentalone or in combination with, for example, an alcohol, acetone, xyleneor glycol (separately or as a mixture). In another embodiment, thecomposition including a dioxolane can be used alone or in combinationwith an alcohol (separately or as a mixture) as a dehydration agentafter a fixation process. Still further, a composition including adioxolane can be used in an infiltration process with, for example, anadditive such as paraffin to improve infiltration of the paraffin in thetissue.

As a clearing agent, a composition including a dioxolane may be used ata concentration of 100 percent (e.g., 100 percent dioxolane composition)or may be combined with another clearing agent or agents such ashydrocarbon clearing agents (e.g., xylene, hexane, mineral oil),hydrocarbon clearing agents with oxygen-based functional groups (e.g.,alcohols (e.g., ethanol), acetates, ethers, acetals, etc.) or mixturesof clearing agents at a lower composition (e.g., 70 percent to 85percent of a dioxolane with the remainder another clearing agent oragents).

The following are example uses of a composition including a dioxolane inpre-analytical processes to treat a tissue taken from a body fordiagnosis.

-   -   1. The use of a dehydrating reagent such as ethanol, methanol,        isopropanol, acetone etc. or combinations of such reagents to        achieve dehydration of the tissues followed by use of a        dioxolane as an independent reagent or as a mixture with other        reagents such as ethanol, xylene, etc. for clearing. Specific        examples include:        -   a) the use of 95 to 100 percent ethanol for dehydration            followed by the use of 1,3-dioxolane for clearing;        -   b) the use of an ethanol-isopropyl alcohol mixture (70:30            v/v) for dehydration followed by the use of 1,3-dioxolane            for clearing;        -   c) the use of acetone-isopropyl alcohol-ethylene glycol            mixture for dehydration followed by the use of 1,3-dioxolane            for clearing;        -   d) the use of 70 percent reagent ethanol (absolute ethanol            denatured with 1 to 10 percent isopropanol and methanol) for            dehydration followed by the use of 1,3-dioxolane for            clearing; and        -   e) the use of 95 to 100 percent reagent ethanol with up to 2            percent acetic acid followed by the use of 1,3-dioxolane for            clearing.    -   2. The use of a 1,3-dioxolane-reagent ethanol mixture (e.g.,        80:20, 85:15, 90:10, 95:5 v/v) for clearing.    -   3. The use of a dioxolane as a part of a dehydrating mixture to        achieve tissue dehydration and again using the dioxolane to        achieve clearing as either a standalone clearing reagent or as a        part of a clearing mixture such as those listed above.        Representative mixtures for dehydration include reagent ethanol        (e.g., ethanol) 1,3-dioxolane mixture (70:30; 50:50 and 30:70        v/v).    -   4. The use of a dioxolane as an additive in paraffin wax (e.g.,        up to 20 percent dioxolane in paraffin) to facilitate        infiltration of the wax into the tissue.

A composition including a dioxolane for a dehydrating, clearing orinfiltration process may be used on conventional tissue processors forconventional processing protocols that generally are several hours longor for short protocols of lesser time (e.g., 60 minutes or less). Acomposition including a dioxolane can also be used in such processes onconventional tissue processors for processing protocols executed atelevated temperatures up to 70° C. reagent processing temperature. Forexample, a composition including a dioxolane with reagent alcohol fortissue dehydration and alone or as part of a mixture for clearing may beperformed at operating temperatures with no added heat to the processingor at elevated reagent temperatures of 60° C. For infiltrationprocesses, a composition including a dioxolane may be combined withparaffin at a temperature on the order of 65-70° C.

A composition including a dioxolane for use as a dehydrating agent, aclearing agent or an infiltration agent is also suitable with microwaveassisted tissue processing for general processing protocols of about 60minutes for regular sized tissues and shorter time periods for smallertissues (biopsy, core, etc.)

EXAMPLES

The following represent specific examples of a use of a compositionincluding a dioxolane in pre-analytic processing.

Example 1

Tissues were grossed and fixed in 10 percent neutral buffered formalinfor 6 to 24 hours. The tissues were then placed in reagent alcohol(90-100 percent) for 30 minutes. The tissues were then placed in1,3-dioxolane for 40 minutes, with microwave processing. Finally, thetissues were infiltrated by placement in paraffin mixed with1,3-dioxolane (5 percent v/v) at about 65° C. for 40 minutes.

Example 2

Tissues were grossed and fixed in 10 percent neutral buffered formalinfor 6 to 24 hours. The tissues were then placed in two consecutivereagent alcohol (95-100 percent) stations for 15 minutes each (or onereagent alcohol station for 30 minutes). The tissues were then placed intwo consecutive 1,3-dioxolane stations for 15 minutes each. Finally, thetissues were infiltrated by placement in two consecutive paraffin (65°C.) stations for 15 minutes each.

Example 3

Tissues were grossed and fixed in 10 percent neutral buffered formalinfor 6 to 24 hours. The tissues were then placed in two consecutivereagent alcohol (95-100 percent) stations for 30 minutes each (or onereagent alcohol station for 30-60 minutes). The tissues were then placedin two consecutive 1,3-dioxolane stations for 30 minutes each. Finally,the tissues were infiltrated by placement in two consecutive paraffin(65° C.) stations for 30 minutes each.

Example 4

In this example, the processing protocol of Example 2 was followed andthe processing was done by microwave assist for the 1,3-dioxolane stepand paraffin steps.

The above examples primarily related to the use of an acetal solvent(e.g., a dioxolane) in treating a biological sample where the treatingis dehydrating, clearing or infiltrating. An acetal solvent can also beused as a fixative.

The building blocks of 1,3-dioxolane, for example, are formaldehyde andethylene glycol or ethylene oxide. 1,3-dioxolane or formal glycol ismanufactured industrially either by condensation reaction offormaldehyde with ethylene glycol in presence of Brönsted or Lewis acidcatalysts or by a reaction of formaldehyde with ethylene oxide inpresence of catalysts such as tin tetrachloride (SnCl₄),tetraethylammonium bromide (CH₃CH₂)₄NBr or toluenesulfonic acid (TsOH).Conversely, formaldehyde can be obtained by cleaving the 1,3-dioxolanemolecule by hydrolysis or other methods.

Various literatures have cited ways to form aldehydes from acetals.Hydrolysis of 1,3-dioxolane reverses the condensation reaction to yieldformaldehyde and ethylene glycol at relatively mild conditions. Thereaction can be carried out by adding hydrochloric acid into the1,3-dioxolane. Varying normality (N) and volume can yield varyingamounts of formaldehyde in the reaction. Use of 6N HCl, for example,offers a mole of formaldehyde for each mole of 1,3-dioxolane in aboutfour hours at room temperature.

Deprotection of acetals and ketals was shown in presence of acetone andindium(III)trifluoromethane-sulfonate (In(OTf)₃) as a catalyst at roomtemperature in a 30 minutes to eight hours duration. The reaction can beaccelerated by use of microwaves at 100° C. to complete the reaction infive to 15 minutes.

A gentle Lewis acid catalyst Er(OTf)₃ was used for chemoselectivecleavage of cyclic acetals at room temperature in wet nitromethane.Similarly, a chemoselective method for cleavage of acetals in wetnitromethane by using catalytic cetium(III)triflate at almost neutral pHoffered high yields in a one to 24 hour period.

Deprotection of acyclic and cyclic acetals in excellent yields withinminutes under neutral conditions in presence of a catalytic amount ofiodine has also been reported.

In one embodiment, a system and process where 1,3-dioxolane is cleavedeither in-situ with one or more tissues placed into it or shortly beforeaddition of a tissue or tissues by means of catalyst, temperature,pressure, addition of water, microwave and combinations thereof isdescribed. A portion of the 1,3-dioxolane will undergo hydrolysis toyield formaldehyde which helps with fixation of the tissue(s) while aremaining portion of the 1,3-dioxolane in the system is available to aidwith processing of the tissue(s) simultaneously by dehydrating andclearing the tissue(s). The described process may be used with thetissues placed through reagent alcohol, ethanol, methanol, isopropylalcohol, acetone or similar dehydrating agents of various concentrationsfor dehydration which will improve tissue clearing.

Representatively, human or animal tissues are collected and placed in asealable container or a reaction chamber containing 1,3-dioxolane. Waterand a few drops of hydrochloric acid are added to the container. Thehydrolysis is carried out in relatively mild or room conditions oftemperature and pressure to yield formaldehyde and ethylene glycol. Theformaldehyde gas obtained in the reaction is dissolved in the water tomake a formalin fixative. The process can be carried out with addedbuffer reagents. The reaction may also be catalyzed by use of Lewisacids, Bronsted acids and other commercial catalysts.

Fixation using an acetal such as 1,3-dioxolane may be performed ontissues that are freshly obtained, previously frozen or previouslydehydrated. Formaldehyde from 1,3-dioxolane can be obtained by differentmethods available in literature. The hydrolysis reaction of1,3-dioxolane can be achieved by different proton donor acid catalysts,for example, hydrochloric acid (HCl), sulfuric acid (H₂SO₄),commercially available acid based catalysts such as toluene sulfonicacid catalysts, and Lewis acids including, but not limited to, iodine,bromine, cetium (III)triflate and indium (III)trifluoromethanesulfonate. The pH of the system can be controlled by using bufferchemicals such as phosphates. Variation in concentration of the acidused in the hydrolysis reaction changes the rate of reaction and yieldof formaldehyde. The ratio of 1,3-dioxolane, deionized (D.I.) water andthe catalyst can be varied to vary a rate of the reaction and to vary ayield of the formaldehyde. The rate of reaction can be changed andaltered by changing the reaction temperature. Higher temperatureincreases the rate of reaction while lower temperature reduces it.Microwave may be used to optimize rate of the reaction.

Example 5

An example of use of dioxolane as a tissue fixative is:

In 79.5 gm. 1,3-dioxolane added is 19.5 gm. D.I. water. Tissues that maybe freshly obtained, previously frozen or dehydrated, are placed intothe mixture of 1,3-dioxolane and water. A few drops (e.g., 2 to 4 drops)of 6N hydrochloric acid are added into the system and the reactionchamber is closed to avoid escape of formaldehyde gas. Mixing isprovided to obtain a homogeneous mixture. The acid catalyzed hydrolysisreaction of the dioxolane yields ethylene glycol and formaldehyde whichis expected to dissolve in the unreacted water. The freshly formedformalin reacts with the proteins in the tissues to achieve fixationwhile the dioxolane helps with clearing of the tissue by removinglipids.

IMPLEMENTATIONS

Implementation 1 is a method including placing a biological sample takenfrom a body into a chamber; adding a composition including an acetalsolvent to the chamber; and fixating the biological sample.

In Implementation 2, the acetal solvent in the method of Implementation1 includes a dioxolane.

In Implementation 3, the acetal solvent in the method of Implementation1 includes 1,3-dioxolane.

In Implementation 4, the composition in the method of Implementation 3includes a mixture including the dioxolane, a catalyst and water.

In Implementation 5, the catalyst in the method of Implementation 4 isan acid.

In Implementation 6, the catalyst in the method of Implementation 4 isan acid based catalyst.

In Implementation 7, the catalyst in the method of Implementation 4 is aLewis acid.

In Implementation 8, after fixing the biological sample, the method ofImplementation 5 includes dehydrating and clearing the biological samplewherein at least a part of at least one of dehydrating and clearingincludes treating the biological sample with a composition including anacetal solvent.

In Implementation 9, treating the biological sample with a compositionincluding an acetal solvent includes clearing the biological sample and,prior to clearing, the method of Implementation 8 includes dehydratingthe fixated biological sample.

In Implementation 10, dehydrating of the method of Implementation 9includes treating the fixed biological sample with a dehydratingcomposition including an alcohol.

In Implementation 11, the dehydrating composition of the method ofImplementation 9 includes at least one of acetone and a glycol.

In Implementation 12, after fixing the biological sample, the method ofImplementation 1 includes dehydrating, clearing the biological sampleand infiltrating the biological sample, and infiltrating the biologicalsample including treating the biological sample with a compositionincluding an acetal solvent.

In Implementation 13, the composition of the method of Implementation 11further includes paraffin.

Implementation 14 is a method including placing a tissue taken from abody into a chamber; and contacting the tissue with a compositionincluding an acetal solvent as a fixating process.

In Implementation 15, the biological sample of the method of any ofImplementations 1-14 includes a tissue.

In Implementation 16, the acetal solvent of the method of Implementation14 includes a dioxolane.

In Implementation 17, the composition in the method of Implementation 15includes a mixture of the dioxolane, water and a catalyst.

In Implementation 18, the catalyst in the method of Implementation 17 isan acid.

In Implementation 19, the composition in the method of Implementation 15includes a dehydrating process, a clearing process and an infiltrationprocess wherein at least one of the dehydrating process, the clearingprocess and the infiltration process includes contacting the tissue withan acetal solvent.

In Implementation 20, the acetal solvent in the at least one of thedehydrating process, the clearing process and the infiltration processin the method of Implementation 19 includes 1,3-dioxolane.

In the description above, for the purposes of explanation, numerousspecific details have been set forth in order to provide a thoroughunderstanding of the embodiments. It will be apparent however, to oneskilled in the art, that one or more other embodiments may be practicedwithout some of these specific details. The particular embodimentsdescribed are not provided to limit the invention but to illustrate it.The scope of the invention is not to be determined by the specificexamples provided above but only by the claims below. In otherinstances, well-known structures, devices, and operations have beenshown in block diagram form or without detail in order to avoidobscuring the understanding of the description. Where consideredappropriate, reference numerals or terminal portions of referencenumerals have been repeated among the figures to indicate correspondingor analogous elements, which may optionally have similarcharacteristics.

It should also be appreciated that reference throughout thisspecification to “one embodiment”, “an embodiment”, “one or moreembodiments”, or “different embodiments”, for example, means that aparticular feature may be included in the practice of the invention.Similarly, it should be appreciated that in the description variousfeatures are sometimes grouped together in a single embodiment, figure,or description thereof for the purpose of streamlining the disclosureand aiding in the understanding of various inventive aspects. Thismethod of disclosure, however, is not to be interpreted as reflecting anintention that the invention requires more features than are expresslyrecited in each claim. Rather, as the following claims reflect,inventive aspects may lie in less than all features of a singledisclosed embodiment. Thus, the claims following the DetailedDescription are hereby expressly incorporated into this DetailedDescription, with each claim standing on its own as a separateembodiment of the invention.

What is claimed is:
 1. A composition for use in a treatment of abiological sample taken from a body prior to containing said biologicalsample with a paraffin, the composition comprising 1,3 dioxolane and asecond agent of a paraffin wax, wherein the 1,3-dioxolane is present inthe composition in an amount of 20 percent by volume or less.